Phthalocyanine formulation and uses thereof

ABSTRACT

The present invention provides a chemiluminescent ink formulation, comprising: a phthalocyanine metal catalyst; a visible dye; and a solvent. The formulation is useful in catalyzing a chemiluminescent reaction, by admixing for example, luminol or isoluminol with an oxidizing agent, a base and the chemiluminescent ink formulation to emit light.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application No. 61/955,743filed Mar. 19, 2014, the teachings of which are hereby incorporated byreference in its entirety for all purposes.

FIELD OF THE INVENTION

This invention relates to a phthalocyanine metal catalyst formulationthat is useful for a pen for marking on membranes (for example, themolecular weight) such as nitrocellulose, PVDF and other membranes.

BACKGROUND OF THE INVENTION

Western blotting is a very useful and common laboratory procedure. Inthe typical procedure, protein mixtures are separated on polyacrylamidegel into bands using an applied electric field. After the proteins areseparated into bands, the separated bands are transferred to a membrane.After transfer, the separated proteins are probed with primary andsecondary antibodies for detection. The detection can be accomplishedvia radioactivity, chemiluminescence, fluorescence, or absorbance. Themost common detection method is chemiluminescence; however, in order todetect multiple analytes simultaneously, fluorescence has recentlygained popularity.

After the separated protein bands are transferred to a membrane, it isoften useful to show the separated bands, or to provide orientation orother experimental information from the membrane. Annotation and/ormarking the position of protein standards for a Western blot membrane istypically done using a Sharpie® or other indelible fine tip felt pen.The film is overlaid and aligned atop the membrane and standardpositions such as a protein ladder are noted on the film. With the useof imagers as a replacement to film, a need to mark the membranedirectly is needed. Currently, other technologies for marking Westernblot membranes utilize phosphorescent (Glow Writer®) or luminescent pens(Advansta®). Due to the various wash steps, it is often difficult tofind suitable types of ink that will withstand the harsh wash steps.

In view of the foregoing, there is a need in the art for a pen to markmembranes and other solid surfaces which will be exposed to biologicalor chemical substrates and procedures. The present invention satisfiesthis and other needs.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a chemiluminescent inkformulation, the chemiluminescent ink formulation comprising:

-   -   a phthalocyanine metal catalyst;    -   optionally a visible dye; and    -   a solvent.

In certain instances, the phthalocyanine metal catalyst has Formula I:

In Formula I, M is a member selected from the group consisting of Ni,Mn, Fe, Co, and Ru. In certain aspects, Mn and Ru are preferred metalsof the phthalocyanine metal catalyst of the present invention.

In Formula I, R¹, R², R³ and R⁴ are each independently selected from thegroup consisting of hydrogen, amino, amido, alkyl, alkenyl, alkoxy,carboxyl, cyano, halo, hydroxyl, sulfonato, phospho, hydroxyalkyl,alkoxyalkyl, aminoalkyl, amidoalkyl, alkylthioalkyl, carboxyalkyl,alkoxycarbonylalkyl, sulfonatoalkyl, alkoxycarbonyl, alkoxyalkyl, asugar residue, a polysaccharide residue, and a PEG; and m, y, z and nare each independently selected from the group consisting of 0, 1, 2, 3and 4.

In another embodiment, the present invention provides a chemiluminescentpen, the chemiluminescent pen comprising:

-   -   a barrel comprising a reservoir for holding a chemiluminescent        ink formulation; and    -   a nib.

In yet another embodiment, the present invention provides a method forcatalyzing a chemiluminescent reaction, the method comprising:

-   -   admixing luminol, isoluminol or a luminol derivative with an        oxidizing agent, a base and    -   a chemiluminescent ink formulation to catalyze a        chemiluminescent reaction and emit light.

These and other aspects, objects and advantages will become moreapparent when read with the drawings and detailed description whichfollow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a pen embodiment of the present invention.

FIGS. 2 A-B illustrate the use of an embodiment of the presentinvention. FIG. 2A shows the use of a ruthenium phthalocyanine metalcatalyst. FIG. 2B shows the use of a manganese phthalocyanine metalcatalyst.

FIGS. 3A-B illustrate the use of an embodiment of the present invention.FIG. 3A shows the imaging of a ruthenium phthalocyanine metal catalystside-by-side with a manganese phthalocyanine metal catalyst on a smallimager. Images were acquired immediately after incubation with substrateon a LI-COR Odyssey® Fc at 2 min exposure. FIG. 3B shows the imaging ofa ruthenium phthalocyanine metal catalyst side-by-side with manganesephthalocyanine metal catalyst on an C-DiGit imager.

FIG. 4 illustrates a schematic diagram of an exemplary use of aChemiluminescent pen of the present invention in Western blotting.

FIGS. 5A-D illustrate the use of an embodiment of the present invention.FIGS. 5A and 5C illustrate the use with a first chemiluminescentsubstrate and FIGS. 5B and 5D illustrate the use of a secondchemiluminescent substrate.

FIG. 6 illustrates the use of an embodiment of the present invention.FIG. 6 shows a pattern similar to that of a pre-stained proteinmolecular weight ladder marked on a membrane.

FIGS. 7A-D illustrates the use of an embodiment of the presentinvention. FIG. 7A shows markings with the Advansta ChemiPen (lanes 1and 2, from left to right) and with an inventive chemiluminescent Pen(lanes 3 and 4, from left to right) on the semi-dry PVDF membrane. FIG.7B shows markings with the Advansta ChemiPen (lanes 5 and 6, from leftto right) and with an inventive chemiluminescent Pen (lanes 7 and 8,from left to right) on the dry PVDF membrane. FIGS. 7C-D show the use onnitrocellulose membrane.

FIG. 8 illustrates the use of an embodiment of the present invention.The image shows the annotated protein ladder in the right lane using thepen of the present invention and different levels of Erk protein.

DETAILED DESCRIPTION I. Definitions

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forexample, an embodiment of a method of imaging that comprises using acompound set forth in claim 1 would include an aspect in which themethod comprises using two or more compounds set forth in claim 1.

The term “about” as used herein to modify a numerical value indicates adefined range around that value. If “X” were the value, “about X” wouldindicate a value from 0.9X to 1.1X, and more preferably, a value from0.95X to 1.05X. Any reference to “about X” specifically indicates atleast the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X,1.03X, 1.04X, and 1.05X. Thus, “about X” is intended to teach andprovide written description support for a claim limitation of, e.g.,“0.98X.”

When the quantity “X” only allows whole-integer values (e.g., “Xcarbons”) and X is at most 15, “about X” indicates from (X−1) to (X+1).In this case, “about X” as used herein specifically indicates at leastthe values X, X−1, and X+1. If X is at least 16, the values of 0.90X and1.10X are rounded to the nearest whole-integer values to define theboundaries of the range.

When the modifier “about” is applied to describe the beginning of anumerical range, it applies to both ends of the range. Thus, “from about700 to 850 nm” is equivalent to “from about 700 nm to about 850 nm.”When “about” is applied to describe the first value of a set of values,it applies to all values in that set. Thus, “about 680, 700, or 750 nm”is equivalent to “about 680 nm, about 700 nm, or about 750 nm.” However,when the modifier “about” is applied to describe only the end of therange or only a later value in the set of values, it applies only tothat value or that end of the range. Thus, the range “about 2 to about10” is the same as “about 2 to about 10,” but the range “2 to about 10”is not.

“Alkanoyl” as used herein includes an alkyl-C(O)— group wherein thealkyl group is as defined herein. Representative alkanoyl groups includeacetyl, ethanoyl, and the like.

“Alkenyl” as used herein includes a straight or branched aliphatichydrocarbon group of 2 to about 15 carbon atoms that contains at leastone carbon-carbon double bond. Preferred alkenyl groups have 2 to about12 carbon atoms. More preferred alkenyl groups contain 2 to about 6carbon atoms. “Lower alkenyl” as used herein includes alkenyl of 2 toabout 6 carbon atoms. Representative alkenyl groups include vinyl,allyl, n-butenyl, 2-butenyl, 3-methylbutenyl, n-pentenyl, heptenyl,octenyl, decenyl, and the like.

“Alkoxy” as used herein includes an alkyl-O— group wherein the alkylgroup is as defined herein. Representative alkoxy groups includemethoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, heptoxy, and the like.

“Alkoxyalkyl” as used herein includes an alkyl-O-alkylene- group whereinalkyl and alkylene are as defined herein. Representative alkoxyalkylgroups include methoxyethyl, ethoxymethyl, n-butoxymethyl andcyclopentylmethyloxyethyl.

“Alkoxycarbonyl” as used herein includes an ester group; i.e., analkyl-O—CO— group wherein alkyl is as defined herein. Representativealkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl,t-butyloxycarbonyl, and the like.

“Alkoxycarbonylalkyl” as used herein includes an alkyl-O—CO-alkylene-group wherein alkyl and alkylene are as defined herein. Representativealkoxycarbonylalkyl include methoxycarbonylmethyl, ethoxycarbonylmethyl,methoxycarbonylethyl, and the like.

“Alkyl” as used herein includes an aliphatic hydrocarbon group, whichmay be straight or branched-chain, having about 1 to about 20 carbonatoms in the chain. Preferred alkyl groups have 1 to about 12 carbonatoms in the chain. More preferred alkyl groups have 1 to 10 or 1 to 6carbon atoms in the chain. “Branched-chain” as used herein includes thatone or more lower alkyl groups such as methyl, ethyl or propyl areattached to a linear alkyl chain. “Lower alkyl” as used herein includes1 to about 6 carbon atoms, preferably 5 or 6 carbon atoms in the chain,which may be straight or branched. Representative alkyl groups includemethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and3-pentyl.

“Alkylsulfonate ester” as used herein includes an alkyl-SO₃— groupwherein the alkyl group is as defined herein. Preferred alkylsulfonateester groups are those wherein the alkyl group is lower alkyl.Representative alkylsulfonate ester groups include mesylate ester (i.e.,methylsulfonate ester).

An “optionally substituted” alkylsulfonate ester includes analkylsulfonate ester as defined herein, wherein the aryl group isadditionally substituted with from 0 to 3 halo, alkyl, aryl, haloalkyl,or haloaryl groups as defined herein. Preferred optionally substitutedalkylsulfonate groups include triflate ester (i.e.,trifluoromethylsulfonate ester).

“Alkylthio” as used herein includes an alkyl-S— group wherein the alkylgroup is as defined herein. Preferred alkylthio groups are those whereinthe alkyl group is lower alkyl. Representative alkylthio groups includemethylthio, ethylthio, isopropylthio, heptylthio, and the like.

“Alkylthioalkyl” as used herein includes an alkylthio-alkylene- groupwherein alkylthio and alkylene are defined herein. Representativealkylthioalkyl groups include methylthiomethyl, ethylthiopropyl,isopropylthioethyl, and the like.

“Amido” as used herein includes a group of formula Y₁Y₂N—C(O)— whereinY₁ and Y₂ are independently hydrogen, alkyl, or alkenyl; or Y₁ and Y₂,together with the nitrogen through which Y₁ and Y₂ are linked, join toform a 4- to 7-membered azaheterocyclyl group (e.g., piperidinyl).Representative amido groups include primary amido (H₂N—C(O)—),methylamido, dimethylamido, diethylamido, and the like. Preferably,“amido” is an —C(O)NRR′ group where R and R′ are members independentlyselected from the group consisting of H and alkyl. More preferably, atleast one of R and R′ is H.

“Amidoalkyl” as used herein includes an amido-alkylene- group whereinamido and alkylene are defined herein. Representative amidoalkyl groupsinclude amidomethyl, amidoethyl, dimethylamidomethyl, and the like.

“Amino” as used herein includes a group of formula Y₁Y₂N— wherein Y₁ andY₂ are independently hydrogen, acyl, aryl, or alkyl; or Y₁ and Y₂,together with the nitrogen through which Y₁ and Y₂ are linked, join toform a 4- to 7-membered azaheterocyclyl group (e.g., piperidinyl).Optionally, when Y₁ and Y₂ are independently hydrogen or alkyl, anadditional substituent can be added to the nitrogen, making a quaternaryammonium ion. Representative amino groups include primary amino (H₂N—),methylamino, dimethylamino, diethylamino, tritylamino, and the like.Preferably, “amino” is an —NRR′ group where R and R′ are membersindependently selected from the group consisting of H and alkyl.Preferably, at least one of R and R′ is H.

“Aminoalkyl” as used herein includes an amino-alkylene- group whereinamino and alkylene are defined herein. Representative aminoalkyl groupsinclude aminomethyl, aminoethyl, dimethylaminomethyl, and the like.

An “optionally substituted” arylsulfonate ester includes anarylsulfonate ester as defined herein, wherein the aryl group isadditionally substituted with from 0 to 3 halo, alkyl, aryl, haloalkyl,or haloaryl groups as defined herein. Preferred optionally substitutedarylsulfonate esters include tosylate ester (i.e., p-tolylsulfonateester).

“Carboxy” and “carboxyl” as used herein include a HOC(O)— group (i.e., acarboxylic acid) or a salt thereof.

“Carboxyalkyl” as used herein includes a HOC(O)-alkylene- group whereinalkylene is defined herein. Representative carboxyalkyls includecarboxymethyl (i.e., HOC(O)CH₂—) and carboxyethyl (i.e., HOC(O)CH₂CH₂—).

“Halo” or “halogen” as used herein include fluoro, chloro, bromo, oriodo.

“Hydroxyalkyl” as used herein includes an alkyl group as defined hereinsubstituted with one or more hydroxy groups. Preferred hydroxyalkylscontain lower alkyl. Representative hydroxyalkyl groups includehydroxymethyl and 2-hydroxyethyl.

“Luminol derivative” as used herein includes an oxidizable substratesuch as 2,3-dihydro-1,4-phthalazinedione, chemiluminescent cyclicdiacylhydrazide, hydroxyaryl cyclic diacylhydrazides and aminoarylcyclic diacylhydrazides. For example, the compoundo-aminophthalhydrazide-N-acetyl-3-D-glucosaminide (luminol-NAG) and4′-(6′-diethylaminobenzofuranyl)phthalhydrazide-N-acetyl-β-D-glucosaminideserve as a masked form of luminol.

“Membrane” as used herein includes the transfer “substrate” to collectproteins in a Western Blot. After gel electrophoresis, proteins aretransferred to a membrane such as nitrocellulose or PVDF, where they arestained with antibodies specific to the target protein.

“PEG” as used herein includes polyethyleneoxide (PEO) polymers assubstituents for the phthalocyanine metal catalyst. Typical PEOmolecular weights include 300 to about 5000.

“Sulfonato” as used herein includes an —SO₃ ⁻ group, preferably balancedby a cation such as H⁺, Na⁺, K⁺, ammonium (NH₄ ⁺), quaternary ammonium(NR₄ ⁺), wherein each R may be the same or different and is alkyl oraryl, and the like.

“Sulfonatoalkyl” as used herein includes a sulfonato-alkylene- groupwherein sulfonato and alkylene are as defined herein. A more preferredembodiment includes alkylene groups having from 2 to 6 carbon atoms, anda most preferred embodiment includes alkylene groups having 2, 3, or 4carbons. Representative sulfonatoalkyls include sulfonatomethyl,3-sulfonatopropyl, 4-sulfonatobutyl, 5-sulfonatopentyl,6-sulfonatohexyl, and the like.

“Sugar residue” or “polysaccharide residue” as used herein includes asugar substituent for attachment to the phthalocyanine metal catalyst.Monosaccharaides include pentoses such as ribose, xylose and arabinoseand hexoses such as glucose, galactose, mannose, idose and gulose. Othermono- and disaccharides such as fructose and sucrose are also sugarresidues. A polysaccharide residue includes dextrans and celluloses. Theresidue is the sugar without a hydrogen attached to an oxygen. Theoxygen such as the anomeric oxygen, is the point of attachment to thephthalocyanine metal catalyst.

II. Embodiments

The present invention provides a composition comprising a catalyticreagent that functions like a peroxidase, such as horseradish peroxidase(HRP), cytochrome C, or the like. The composition comprising a catalyticreagent produces chemiluminescent light when exposed to achemiluminescent substrate. In certain instances, the compositioncomprises at least one visible dye. The visible dye allows for ease ofidentification of the target such as in an annotated or marked surface.After marking the surface, the visible dye allows for the targetidentification prior to, or after exposure to a chemiluminescentsubstrate.

As such, in one embodiment, the present invention provides achemiluminescent ink formulation, the chemiluminescent ink formulationcomprising:

-   -   a phthalocyanine metal catalyst;    -   optionally a visible dye; and    -   a solvent.

In certain instances, the phthalocyanine metal catalyst has Formula I:

In Formula I, M is a member selected from the group consisting of Ni,Mn, Fe, Co, and Ru. In certain aspects, Mn and Ru are preferred metalsfor the phthalocyanine metal catalyst of the present invention.

In Formula I, R¹, R², R³ and R⁴ are each independently selected from thegroup consisting of hydrogen, amino, amido, alkyl, alkenyl, alkoxy,carboxyl, cyano, halo, hydroxyl, sulfonato, phospho, hydroxyalkyl,alkoxyalkyl, aminoalkyl, amidoalkyl, alkylthioalkyl, carboxyalkyl,alkoxycarbonylalkyl, sulfonatoalkyl, alkoxycarbonyl, alkoxyalkyl, asugar residue, a polysaccharide residue, and a PEG; and m, y, z and nare each independently selected from the group consisting of 0, 1, 2, 3and 4.

In one aspect, R¹, R², R³ and R⁴ are each independently hydrogen oralkoxy. In one aspect, R¹, R², R³ and R⁴ are each independently alkoxyand m, y, z and n are each 2. In one aspect, the alkoxy group is a C₁-C₆alkoxy such as C₁, C₂, C₃, C₄, C₅ or C₆ alkoxy (e.g. propoxy, butoxy, orpentoxy group).

In certain instances, phththalocyanine dyes and their metal complexeshave limited solubility in solvents. However, various substituents atR¹, R², R³, and R⁴, can improve the dye's solubility in a solvent orsolution as these substitutions can reduce aggregation.

The metal can be inserted into the core during the synthesis or insertedafter the dye formation. In either case, the metal complexes havesimilar catalytic activity.

To provide visible orientation and confirmation that the catalyst hasbeen applied, a visible dye is optionally included in thechemiluminescent “ink” formulation. The inclusion of a visible dye isadvantageous, but not required for an application using thechemiluminescent ink formulation. Suitable visible dyes that can be usedinclude, but are not limited to, the dyes listed in Table 1. A preferreddye is Nile Blue A such as Nile Blue A perchlorate, which is especiallysuitable.

TABLE 1 Preferred Visible Dye for use in the Chemiluminescent pen. InkSolvent Mix BA:Toluene:IPA Visible Dye Source Cat. No. (%) Solvent GreenAldrich 211982 0:50:50 Sudan Blue Aldrich 306436 0:30:70 Safranin SigmaS884 0:30:70 1- Sigma M28200-5G 20:0:80 (methylamino)anthraquinone15:15:70 10:20:70 100:0:0 100:0:0 100:0:0 100:0:0 30:0:70 OBPC (nonmetalated) Sigma 383805 0:30:70 Nile Blue A Perchlorate Aldrich 370880:30:70 BA: 4-methoxybenzyl alcohol, Sigma, Cat. No. 136905-100G,Lot#MKBF6704V IPA: Isopropyl Alcohol Catalyst: OBPC + Mn, NB Ref 672-199

The chemiluminescent ink formulation typically includes a solvent. Asuitable solvent for the ink formulation of the present invention is onethat dissolves the catalyst and visible dye. In addition to dissolvingthe catalyst and the visible dye another consideration is the surfacetype wherein the chemiluminescent ink formulation will be applied. Forexample, membrane surfaces typically used in a Western blot or similarapplication includes nitrocellulose, PVDF, and the like. Certainsolvents may affect these surfaces.

In addition to compatibility with membrane surfaces, the solvent mayalso be compatible with the other chemiluminescent ink formulationcomponents. In certain applications, the solvent can be dissolved in awashing buffer(s) and the catalyst is deposited on the markedposition(s) of the membrane. The ideal solvents include, but are notlimited to, water, alcohols, esters, amines, amides, hydrocarbons,halogenated hydrocarbons, ketones, organic oxides or mixture thereof.

In certain instances, an alcohol is used such as aliphatic or aromaticalcohol. Typical alcohols include, but are not limited to, methanol,ethyl alcohol, propanol, and isopropyl alcohol, butanol, pentanol andthe like. Aromatic alcohols include phenols, substituted phenols, benzylalcohol, 4-methoxy benzyl alcohol, phenylethanol, phenoxyethanol, andthe like. Other solvents include glycols, polyethylene glycols,polypropylene glycols, glycerol, esters such as ethyl acetate, butylacetate, benzyl benzoate, ethers such as alkylene glycol alkyl etherssuch as dipropylene glycol monomethyl ether, di-ethylene glycolmono-butyl ether, ketones such as acetone, methyl ethyl ketone, cycliccarbonates such as propylene carbonate, ethylene carbonate, aromaticand/or aliphatic hydrocarbons, vegetable or synthetic oils, DMF,dimethylacetamide, n-alkylpyrrolidones such as methylpyrrolidone,n-butylpyrrolidone or n-octylpyrrolidone, N-methylpyrrolidone,2-pyrrolidone, 2,2-dimethyl-4-oxy-methylene-1,3-dioxolane and glycerolformal. Multiple solvents can be used. In certain aspects, the followingsolvents are used: water/aqueous buffer; methanol, ethanol, 1-pentanol,1-butanol, 2-propanol, toluene, xylene, 4-methoxy benzyl alcohol.

In a chemiluminescent ink formulation application the solvent isdissolved entirely or partially in washing buffer(s) and the catalyst isdeposited on the membrane. The catalytic property of phthalocyaninemetal catalyst is ideal for this application.

A phthalocyanine metal catalyst is stable throughout the Western blotprocess. Metal complexed phthalocyanine metal catalysts produce lightwith a wide variety of substrates. Commercial substrates with high andlow sensitivity can be used. For example, Thermo Scientific™SuperSignal™ West Dura Substrate, which is a luminol-based HRP substratecan be used. In addition, Thermo Scientific™ SuperSignal™ West PicoChemiluminescent Substrate can also be used. Those skilled in the artwill know if other commercial HRP substrates that can be used.

The phthalocyanine metal catalyst is admixed with a substrate. Thesubstrate is typically luminol, isoluminol or luminol derivative,together with an oxidizing agent, and a base. The oxidizing agent can befor example, a perborate or a peroxide. In certain instances, thesubstrate further comprises an enhancer. The enhancer is selected fromthe group consisting of sodium phenothiazine-10-yl-propylsulfonate(PTA), 2,2′Azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS),4-(1,2,4-triazol-1-yl)phenol, 2-(1H-imidazole-2-yl)pyridine,4-dimethylaminopyridine (DMAP), an azine, phenothiazine, phenoxazine anda combination thereof. Other enhancers are disclosed in U.S. Pat. Nos.5,171,668, 6,432,662, and 7,803,573 the disclosures of which are herebyincorporated by reference.

In certain aspects, water solubility of the phthalocyanine metalcatalyst is useful in determining suitability for use in achemiluminescent ink formulation application. For example, a complexwhich is too hydrophilic will be removed by washing buffer and may causethe membrane to have high background.

In certain instances, the chemiluminescent ink formulation is disposedor filled in a writing implement such as “a pen” having a barrelcomprising a reservoir. The chemiluminescent pen or “chemi-pen” hasutility for marking the positions of standards or other annotations onimmunoassays such as Western blot membranes and similar membranesurfaces which are exposed to a chemiluminescent substrate. Thechemiluminescent pen can be used to mark experimental information suchas a date, a name, a lane, a label, a lot number, a sample number andother appropriate information that is valuable for tracking andcross-referencing data in a scientific or diagnostic laboratory.

Prior to the advent of the present invention, annotations and/or markingthe position of protein standards on a Western blot membrane were doneusing a Sharpie® or other indelible fine tip felt pen. One advantage ofthe chemiluminescent pen is that annotations are visible to the eye andthe light produced when exposed to a chemiluminescent substrate is thatthe annotations are captured simultaneously with any chemiluminescentdetection imaging system or film. In addition to a simple CCD imager, noother detector or camera is needed to visualize the visible dye.

In one aspect, the catalyst used to indicate the positions ofpre-stained protein marker bands or other annotations on a Western blotmembrane is applied at very low concentration such that it cannot beseen visually.

The ratio of the phthalocyanine metal catalyst to visible dye in achemiluminescent ink formulation is about 1:10 w/w to about 10:1 w/w. Incertain instances, the phthalocyanine metal catalyst to visible dyeratio is about 1:1; 1:2; 1:3; 1:4; 1:5; 1:6; 1:7; 1:8; 1:9 or 1:10 w/w.In still other instances, the phthalocyanine metal catalyst to visibledye ratio is about 10:1; 9:1; 8:1; 7:1; 6:1; 5:1; 4:1; 3:1; 2:1 or 1:1w/w. In still other instances, the phthalocyanine metal catalyst tovisible dye ratio is about 1:1 to about 1:3 w/w.

The amount of solvent in the formulation will depend on thephthalocyanine metal catalyst and the visible dye used. Typically, about1 mL to 500 mL can be used. In other instances, about 1 mL to 25 mL,such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25 mL of solvent is used.

In one embodiment, the present invention provides a chemiluminescentpen, the chemiluminescent pen comprising:

-   -   a barrel comprising a reservoir for holding a chemiluminescent        ink formulation; and    -   a nib.

In certain instances, the reservoir further comprises a fiber or fillerto adsorb the chemiluminescent ink formulation.

FIG. 1 is one embodiment of a pen 100 of the present invention. As showntherein, the pen 100 has a barrel 110 comprising a reservoir for holdingthe chemiluminescent ink formulation. The barrel 110 may have fiber,filler or fabric contained therein to slowly release thechemiluminescent ink formulation adsorbed thereto. The ink can beadsorbed on fiber or filler and thereafter released. The ink orchemiluminescent ink formulation can be used to fill a reservoir orbarrel. The barrel holds a reservoir and preferably ends in a nib 121 orpoint to dispense the liquid ink. The nib or point 121 can be fine tonote protein marker-bands on a membrane that may be separated by only afew millimeters. The barrel containing the reservoir components arepreferably compatible with solvents chosen for the ink formulation. Inoperation, the ink flows from the reservoir to a nib at a rate thatallows a fine line on the membrane without spreading. In certainaspects, the barrel further comprises a cap 131. The cap is useful forclosing the nib.

In certain other applications, a reservoir is used without fiber so theink can flow quickly to the nib and thereafter to the membrane. To limitevaporation that results in the pen going dry, air tight seals or a capcan be used. The barrel can be loaded with about 1 mL to about 20 mL ofink formulation, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20 mL of ink. The ink can be placed in apre-assembled pen and then capped.

The chemiluminescent pen allows for drawing or marking the transfermembranes in Western blots or other immunoassays. In addition,pre-stained protein standards can be used, and annotated. Thechemiluminescent ink formulation adsorbs to nitrocellulose and PVDFmembranes and reacts with peroxidase (e.g., HRP) substrates to producechemiluminescence. The chemiluminescence can thereafter be detected.

The chemiluminescent ink formulation can be disposed or filled in avariety of reservoirs for different types of writing or printing such asink-jet cartridges.

In certain aspects, the present invention provides a method forcatalyzing a chemiluminescent reaction, the method comprising:

-   -   admixing luminol, isoluminol or luminol derivatives with an        oxidizing agent, a base and    -   a chemiluminescent ink formulation to catalyze a        chemiluminescent reaction and emit light.

The signal duration of the catalytic component of the chemiluminescentink when exposed to a chemiluminescent substrate is an advantageousfeature of the methods disclosed herein. Multiple phthalocyanine dyese.g. phthalocyanine-3,4′,4″,4′″-tetrasulfonic acid and1,4,8,11,15,18,22,25 Octabutoxy-29H,31H-phthalocyanine (“OBPC”) can becomplexed with a variety of metals (e.g. Fe, Co, Ni, Mn, Ru, and thelike) for properties such as early signal intensity and total signalduration. The chemiluminescent pen can be used with low as well as highsensitivity chemiluminescent substrates with short or long duration. Toavoid overexposure of film or saturated digital images, the signalshould be intense in the short run and decrease over the long run. OBPCcomplexed with manganese is a preferred phthalocyanine metal catalyst ofthe present invention because of these properties.

Commercial substrates with high and low sensitivity can be used. Forexample, Thermo Scientific™ SuperSignal™ West Dura Substrate, which is aluminol-based HRP substrate can be used. In addition, Thermo Scientific™SuperSignal™ West Pico Chemiluminescent Substrate can also be used.Other commercial HRP substrates can also be used.

The phthalocyanine metal catalyst is admixed with a substrate. Thesubstrate is typically luminol or isoluminol, together with an oxidizingagent, and a base. The oxidizing agent can be for example, a perborateor a peroxide. In certain instances, the substrate further comprises anenhancer. The enhancer is selected from the group consisting of sodiumphenothiazine-10-yl-propylsulfonate (PTA),2,2′Azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS),4-(1,2,4-triazol-1-yl)phenol, 2-(1H-imidazole-2-yl)pyridine,dimethylaminopyridine (DMAP), an azine, phenothiazine, phenoxazine and acombination thereof. Other enhancers are disclosed in U.S. Pat. Nos.5,171,668, 6,432,662, and 7,803,573 the disclosures of which are herebyincorporated by reference.

In certain aspects, the present invention provides a chemiluminescencereaction method, the method comprising:

-   -   (i) contacting chemiluminescent ink formulation with a reaction        mixture comprising luminol or isoluminol, an oxidant and a base        to emit light;    -   (ii) detecting the emitted light; and    -   (iii) optionally correlating the emitted light to the        concentration of luminol, isoluminol or luminol derivative.

In certain aspects, the correlation can be used with a standard curve toascertain the concentration of unknowns.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1. Synthesis of manganese1,4,8,11,15,18,22,25-Octabutoxy-29H,31H-phthalocyanine

1,4,8,11,15,18,22,25-Octabutoxy-29H,31H-phthalocyanine (2 g, 1.8 mmol)and manganese(II) acetate tetrahydrate (2.2 g, 9.0 mmol) were heated at60° C. in 1-butanol (100 mL) for 45 minutes. The reaction was cooled andthen excess salts were removed by aqueous extractions. The organic layerwas then dried over anhydrous sodium sulfate, filtered and dried to adark red powder (2 g, 97%). The product was characterized using UV-VISand light emission assays.

Example 2. Synthesis of Rutheniumphthalocyanine-3,4′,4″,4′″-tetrasulfonic acid

Phthalocyanine tetrasodium salt (1 gm) and ruthenium(III) chloridetrihydrate were mixed in 20 mL of water and refluxed for 2 hours. Thewater was removed and separated by flash reverse C-18. The final productwas 0.85 g of a green solid.

Example 3. Synthesis of manganesephthalocyanine-3,4′,4″,4′″-tetrasulfonic acid

Manganese phthalocyanine-3,4′,4″,4′″-tetrasulfonic acid was preparedsimilarly according to procedure described in Example 2 and manganesechloride tetrahydrate was used in lieu of ruthenium(III) chloridetrihydrate.

Example 4. Formulation of a Chemiluminescent Ink

To 1.5 g of 1,4,8,11,15,18,22,25-Octabutoxy-Phthalocyanine (OBPC)prepared as in Example 1, 3.0 g of Nile Blue A perchlorate (commerciallypurchased from Aldrich) is admixed. The structure of Nile Blue Aperchlorate is shown below.

The mixture is sonicated overnight in 1 liter of a 30:70 mixture oftoluene and isopropyl alcohol. The mixture is stirred continuously whilereservoirs are loaded to yield a phthalocyanine ink formulation of thepresent invention.

Example 5. Dot Blot Test of Catalytic Activity on a Western BlotMembrane

The catalytic activity of the reagents rutheniumphthalocyanine-3,4′,4″,4′″-tetrasulfonic acid 210 and manganesephthalocyanine-3,4′,4″,4′″-tetrasulfonic acid 255 were tested using aseries of dot blots (FIGS. 2A and 2B). Both reagents were made as asaturated solution and a five-fold serial dilution of each was prepared.The samples were dotted neatly and in triplicate on nitrocellulosemembrane (NCM). The membranes were washed with PBS and developed for 5minutes with Thermo™ SuperSignal™ West Dura Chemiluminescent Substrate.Images were acquired immediately after incubation with substrate onLI-COR Odyssey Fc at 2 min exposure. The catalytic activity was robustfor both compounds. No signal was detected in the blank spots 231. FIG.2A shows that the ruthenium phthalocyanine metal catalyst was depositedat a high concentration 217, a medium concentration 221 and a lowconcentration 224. FIG. 2B shows that the manganese phthalocyanine metalcatalyst was deposited at a high concentration 261, a mediumconcentration 271 and a low concentration 282. The blank 231 containedno catalytic reagent. For both compounds the chemiluminescence washighest at the highest concentration (217 and 261) deposited.Accordingly, the signals were decreased at the lower levels of thereagent.

Example 6. Slot Blot Test of Catalytic Activity on a Membrane

A saturated solution of ruthenium (III)phthalocyanine-3,4′,4″,4′″-tetrasulfonic acid 310 and manganesephthalocyanine-3,4′,4″,4′″-tetrasulfonic acid 320 in ultrapure waterwere painted onto nitrocellulose membranes to mimic the pattern of apre-stained protein molecular weight marker. The membranes were washedwith PBS and developed for 5 minutes with Thermo™ SuperSignal™ West DuraChemiluminescent Substrate.

Images were acquired immediately after incubation with substrate on aLI-COR Odyssey Fc at 2 min exposure (FIG. 3A). The membrane depositedwith ruthenium (III) phthalocyanine-3,4′,4″,4′″-tetrasulfonic acid 310produced a robust signal that resembled a protein molecular weightladder with a highest molecular weight standard 312 and a low molecularweight standard 317. The membrane deposited with manganesephthalocyanine-3,4′,4″,4′″-tetrasulfonic acid 320 also produced a robustsignal that resembled a protein molecular weight ladder with a highestmolecular weight standard 321 and a low molecular weight standard 325.

In a parallel experiment, images of the ruthenium phthalocyanine metalcatalyst reagent and the manganese phthalocyanine metal catalyst reagentwere acquired after a 1 hour exposure with substrate on a C-DiGitscanner (FIG. 3B). The ruthenium phthalocyanine metal catalyst reagent350 generated a pattern that mimicked a protein ladder with a series ofmolecular weights ranging from highest 351 to lowest molecular weight357. The manganese phthalocyanine metal catalyst reagent 370 generated asimilar pattern of molecular weights ranging from highest 372 to lowestmolecular weight 377. The robust signal was stable for greater than 1hour at ambient temperatures. The slot blots demonstrate that thecatalyst reagents are useful for marking membranes.

Example 7. Using the Chemiluminescent Pen in a Western Blot

This example provides a diagram of an exemplary use of thechemiluminescent pen described herein. In particular, the example showsthe use of the pen in Western blotting (FIG. 4). A sample such as a celllysate sample is run on a polyacrylamide gel and the proteins areseparated by gel electrophoresis 410. The proteins are transferred fromthe gel onto a membrane, such as a nitrocellulose membrane, PVDFmembrane or nylon membrane, using standard wet or semi-dry transferconditions 415. The transferred membrane contains the proteins from thegel including the protein molecular weight standard ladder 421. Thechemiluminescent pen 427 is used to deposit the chemiluminescent inkonto the membrane, for example, to annotate a visible molecular weightladder 425. The pen can be used to label or mark the membrane. In someembodiments, the chemiluminescent ink includes a visible dye thatindicates the chemiluminescent markings on the membrane. The membrane isblocked 435 with a blocking solution such as 5% skim milk or BSAsolution to prevent non-specific background binding of the primary orsecondary antibodies to the membrane. The membrane 450 is incubated withprimary antibodies to the protein(s) of interest. The membrane isincubated with a secondary antibody labeled with an enzyme, such ashorseradish peroxidase (HRP) or alkaline phosphatase (AP) 456. Achemiluminescent substrate is incubated with the membrane 470. Emittedlight from the chemiluminescent reaction is detected using an imagingsystem or X-ray film 490. For example, the protein ladder that wasannotated using the chemiluminescent pen is visible on the film.

Example 8. Slot Blot Test of Catalytic Activity on the Membrane

This example illustrates the use of a chemiluminescent ink withsubstrates on a slot blot membrane (FIG. 5A-D). Manganese1,4,8,11,15,18,22,25-Octabutoxy-29H,31H-phthalocyanine was made at aconcentration of 1 mg/ml solution in 30/70 toluene isopropanol. Thesolution was tested similarly to the phthalocyanine metal catalystreagent of Example 6. The chemiluminescent ink was deposited on fournitrocellulose membranes, such that each membrane contained two columnsof six horizontal lines. The membranes were developed with either theThermo Scientific TmSuperSignal™ Pico chemiluminescent substrate or theThermo Scientific™ SuperSignal™ West Dura chemiluminescent substrate andimaged with either a LI-COR Odyssey Fc or a LI-COR C-DiGit scanner.

Membranes developed with Thermo Scientific™ SuperSignal™ Pico are shownin FIGS. 5A and 5C. Images acquired on a LI-COR Odyssey Fc showeddetectable signals, e.g., 512, 517, 521 and 525. Images acquired on aLI-COR C-DiGit scanner also showed robust signals, e.g., 542, 547, 531and 535.

Membranes developed with Thermo Scientific™ SuperSignal™ West Dura areshown in FIGS. 5B and 5D. The chemiluminescent ink produced robustmarker lines, e.g., 551, 557, 572, and 577, as imaged by a LI-COROdyssey Fc. Bold marker lines, e.g., 561, 567, 582, and 587, were alsovisible using a LI-COR C-DiGit scanner.

Example 9. Storage Stability of Phthalocyanine Metal Catalyst

Manganese phthalocyanine-3,4′,4″,4′″-tetrasulfonic acid in ultrapurewater was marked onto a nitrocellulose membrane. The chemiluminescentink was used to pen the terms “LI-COR” and “Exp4.” A pattern similar tothat of a pre-stained protein molecular weight ladder was also marked onthe membrane. The membrane was stored on the bench top for 1 week atambient temperature exposed to light. After 1 week, Thermo™ SuperSignal™West Dura substrate was added and the membrane was imaged on a LI-COROdyssey® Fc (FIG. 6). The marking on the membrane were clearly visibleafter the chemiluminescent reaction. Thus, the phthalocyanine metalcatalyst reagent was stable even after a one-week exposure to ambienttemperature and light.

Example 10. Comparison of Chemiluminescent Pen and Advansta ChemiPenSignals on PVDF and Nitrocellulose Membranes

This example compares the performance of the chemiluminescent pendescribed herein to the Advansta ChemiPen on PVDF and nitrocellulosemembranes.

The PVDF membranes were wet with methanol and rinsed with 1×PBS. ExcessPBS was drained from the membranes (semi-Dry) and the membranes weremarked with either the Advansta ChemiPen (Advansta, Menlo Park, Calif.)or the chemiluminescent Pen (LI-COR).

The nitrocellulose membranes were wet with 1×PBS. Excess PBS was drainedfrom the membranes (semi-dry) and the membranes were marked with eitherthe Advansta ChemiPen or LI-COR's chemiluminescent Pen.

PVDF and nitrocellulose membranes were marked multiple times (Heavy=overand over, back and forth) or once (Light=single pen stroke). The markingprocess was repeated on dry (not pre-wet) membranes. The membranes wereincubated with 5% skim milk in 1×PBS and washed extensively with PBST(PBS plus 0.1% Tween 20) followed by PBS. Thermo™ SuperSignal™ West Durasubstrate was added and the membranes were imaged on a LI-COR Odyssey®Fc for 2 min immediately after substrate addition (Time 0) or after onehour (Time 1 hr) following substrate addition.

FIG. 7A shows markings with the Advansta ChemiPen (lanes 1 and 2) andwith LI-COR's chemiluminescent Pen (lanes 3 and 4) on the semi-dry PVDFmembrane. FIG. 7B shows markings with the Advansta ChemiPen (lanes 5 and6) and with LI-COR's chemiluminescent Pen (lanes 7 and 8) on the dryPVDF membrane. LI-COR's pen produced darker lines on the PVDF membranecompared to the Advansta ChemiPen in all conditions tested.

For the nitrocellulose membrane, the pen markers were darker when themembrane was imaged immediately after the substrate was incubated withthe membrane (FIGS. 7C and 7D). On semi-dry nitrocellulose membraneLI-COR's chemiluminescent ink generated more robust lines (lanes 3 and4) compared to Advansta's pen (lanes 1 and 2). On dry nitrocellulose,both pens produced markings of similar intensity (FIG. 7D).

Example 11. Chemiluminescent Pen Used to Mark the Protein Standards on aWestern Blot

This example illustrates the use of the chemiluminescent pen on aWestern blot for Erk protein. Serial dilutions of NIH/3T3 cell lysatewere separated by SDS-PAGE and transferred to a nitrocellulose membrane.After transfer, the positions of the pre-stained protein molecularweight marker (“Marker”) bands were annotated using the of the presentinvention. The membrane was subsequently blocked with 5% skim milk inPBS and further processed according to a standard Western blottingmethod. The primary antibody was an anti-Erk monoclonal antibody and thesecondary antibody was a horseradish peroxidase conjugated goatanti-mouse secondary antibody. The membrane was washed with PBST andPBS. Thermo™ SuperSignal™ West Dura substrate was added and the membranewas imaged on a LI-COR Odyssey® Fc for 2 min immediately after substrateaddition. The image (FIG. 8) shows the annotated protein ladder in theright lane and different levels of Erk protein.

Example 12. The Synthesis of2,9,16,23-Tetra-Tert-Butyl-29H,31H-Phthalocyanine Manganese

2,9,16,23-Tetra-tert-butyl-29H,31H-phthalocyanine manganese is madesimilarly as described in Example 1 except by using2,9,16,23-Tetra-tert-butyl-29H,31H-phthalocyanine (Aldrich, catalog#423157) as starting material.

Example 13. The Synthesis of Tetra-Tetraethyleneglycol Monomethyl EtherPhthalocyanines Manganese

A mixture of 4-(monomethyl ether Tetraethyleneglycol phthalonitrile(0.52 g, 1.79 mmol), and MnCl₂.4H₂O (1.08 g, 5.38 mmol) in n-pentanol(20 mL) is heated to 100° C., and then a small amount of DBU (1 mL) isadded. The mixture is stirred at 140-150° C. for 24 h. After a briefcooling, the volatiles are removed under reduced pressure. The residueis dissolved in CHCl₃ (150 mL) and then filtered to remove part of theMnPc formed. The filtrate is collected and evaporated to dryness invacuo. The residue is purified by silica gel column chromatography usingCHCl₃ and then CHCl₃/MeOH (100:1 v/v) as the eluents. The crude productis purified by size exclusion chromatography using THF as the eluent,followed by recrystallization from a mixture of CHCl₃ and hexane to givea green solid.

Example 14. The synthesis of2(3),9(10),16(17),23(24)-Tetrakis(α/β-D-galactopyranosyl-oxy)Phthalocyaninato Manganese

To 4-(D-glucopyranosyloxy)-1,2-Benzenedicarbonitrile (3.25 g, 1.06 mmol)dissolved in a mixture of 2-(dimethylamino)ethanol (10 mL) and n-butanol(5 mL) is added manganese chloride tetrahydrate (2.33 g, 10 mmol). Themixture is stirred under argon for 24 h at 100° C. and concentrated invacuo. The residue is then dissolved in a minimal amount of H₂O andcrude product was precipitated by addition of acetone. The solid isfiltered, redissolved in a minimal amount of H₂O, precipitated a secondtime by addition of acetone, and collected by filtration. Purificationwas achieved by flash RP(C18) chromatography (H₂O-MeCN, 10:1) to afford1 as a green solid (1.40 g, 10%).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A chemiluminescent ink formulation adsorbed to amembrane, the chemiluminescent ink formulation comprising: aphthalocyanine metal catalyst, wherein the metal is a member selectedfrom the group consisting of Ni, Mn, Fe, Co, and Ru; a visible dye,other than the phthalocyanine metal catalyst; and a solvent, wherein theformulation is adsorbed to the membrane which membrane is a memberselected from the group consisting of nitrocellulose, PVDF, and nylon.2. The chemiluminescent ink formulation of claim 1, wherein thephthalocyanine metal catalyst has the formula:

wherein M is a member selected from the group consisting of Ni, Mn, Fe,Co, and Ru; R¹, R², R³ and R⁴ are each independently selected from thegroup consisting of hydrogen, amino, amido, alkyl, alkenyl, alkoxy,carboxyl, cyano, halo, hydroxyl, sulfonato, phospho, hydroxyalkyl,alkoxyalkyl, aminoalkyl, amidoalkyl, alkylthioalkyl, carboxyalkyl,alkoxycarbonylalkyl, sulfonatoalkyl, alkoxycarbonyl, and alkoxyalkyl, asugar residue, a polysaccharide residue, and a PEG; and m, y, z and nare each independently selected from the group consisting of 0, 1, 2, 3and
 4. 3. The chemiluminescent ink formulation of claim 2, wherein M isMn.
 4. The chemiluminescent ink formulation of claim 2, wherein M is Ru.5. The chemiluminescent ink formulation of claim 2, wherein R¹, R², R³and R⁴ are each alkoxy.
 6. The chemiluminescent ink formulation of claim5, wherein m, y, z and n are each
 2. 7. The chemiluminescent inkformulation of claim 1, wherein the visible dye is a member selectedfrom the group consisting of Solvent Green, Sudan Blue, Safranin,1-(methylamino)anthraquinone and Nile Blue A.
 8. The chemiluminescentink formulation of claim 7, wherein the visible dye is Nile Blue A. 9.The chemiluminescent ink formulation of claim 1, wherein the solvent isa member selected from the group consisting of water, an alcohol, anester, an amine, an amide, a hydrocarbon, a halogenated hydrocarbon, aketone, an organic oxide and a mixture thereof.
 10. The chemiluminescentink formulation of claim 9, wherein the solvent is a mixture thereof ofan alcohol and a hydrocarbon.
 11. A chemilumine scent ink formulation ofclaim 1, wherein the formulation prior to being adsorbed on the membraneis disposed or filled in a reservoir.
 12. A chemiluminescent inkformulation of claim 11, wherein said reservoir comprises a fiber orfiller to adsorb the chemiluminescent ink formulation.
 13. A method forcatalyzing a chemiluminescent reaction with a chemiluminescent inkformulation absorbed to a membrane, said method comprising: Admixingluminol, isoluminol or luminol derivative with an oxidizing agent, abase and the chemiluminescent ink formulation absorbed to the membrane,wherein the chemiluminescent ink formulation comprises: A phthalocyaninemetal catalyst, wherein the metal is a member selected from the groupconsisting of Ni, Mn, Fe, Co, and Ru; a visible dye, other than thephthalocyanine metal catalyst; and a solvent, and wherein the membraneis a member selected from the group consisting of nitrocellulose, PVDF,and nylon, to catalyze a chemiluminescent reaction and emit light. 14.The method of claim 13, wherein the phthalocyanine metal catalyst hasthe formula:

wherein M is a member selected from the group consisting of Ni, Mn, Fe,Co, and Ru.
 15. The method of claim 14, wherein the phthalocyanine metalcatalyst has the formula:

wherein M is a member selected from the group consisting of Ni, Mn, Fe,Co, and Ru; R¹, R², R³ and R⁴ are each independently selected from thegroup consisting of hydrogen, amino, amido, alkyl, alkenyl, alkoxy,carboxyl, cyano, halo, hydroxyl, sulfonato, phospho, hydroxyalkyl,alkoxyalkyl, aminoalkyl, amidoalkyl, alkylthioalkyl, carboxyalkyl,alkoxycarbonylalkyl, sulfonatoalkyl, alkoxycarbonyl, alkoxyalkyl, asugar, a polysaccharide, and a PEG; and m, y, z and n are eachindependently selected from the group consisting of 0, 1, 2, 3 and 4.16. The method of claim 13, wherein the oxidizing agent is a memberselected from the group consisting of perborate and peroxide.
 17. Achemiluminescence reaction method with a chemiluminescent inkformulation adsorbed to a membrane, said method comprising: (i)contacting chemilumine scent ink formulation adsorbed to the membrane,wherein the chemiluminescent ink formulation comprises: a phthalocyaninemetal catalyst, wherein the metal is a member selected from the groupconsisting of Ni, Mn, Fe, Co, and Ru; a visible dye, other than thephthalocyanine metal catalyst; and a solvent, wherein the membrane is amember selected from the group consisting of nitrocellulose, PVDF, andnylon, with a reaction mixture comprising luminol, isoluminol or aluminol derivative, an oxidant and a base to emit light; (ii) detectingthe emitted light; and (iii) optionally correlating the emitted light tothe concentration of luminol or isoluminol.
 18. The chemiluminescencereaction method of claim 17, wherein the phthalocyanine metal catalysthas the formula:

wherein M is a member selected from the group consisting of Ni, Mn, Fe,Co, and Ru; R¹, R², R³ and R⁴ are each independently selected from thegroup consisting of hydrogen, amino, amido, alkyl, alkenyl, alkoxy,carboxyl, cyano, halo, hydroxyl, sulfonato, phospho, hydroxyalkyl,alkoxyalkyl, aminoalkyl, amidoalkyl, alkylthioalkyl, carboxyalkyl,alkoxycarbonylalkyl, sulfonatoalkyl, alkoxycarbonyl, alkoxyalkyl, asugar residue, a polysaccharide residue, and a PEG; and; and m, y, z andn are each independently selected from the group consisting of 0, 1, 2,3 and 4.